Wound healing dressing for enhancing fibrocyte formation

ABSTRACT

The present invention relates to the ability of SAP to suppress the differentiation of monocytes into fibrocytes. It also relates to the ability of IL-4 and IL-3 to enhance the differentiation of monocytes into fibrocytes. Methods and compositions for binding SAP, decreasing SAP levels and suppressing SAP activity are provided. Methods of using, inter alia, CPHPC, the 4,6-pyruvate acetyl of beta-D-galactopyranose, ethanolamines, high EEO agarose, IL-4, and IL-13, and anti-SAP antibodies and fragments thereof to increase monocyte differentiation into fibrocytes are provided. These methods are useful in a variety of applications, including wound healing. Wound dressings are also provided. Finally, the invention may include assays for detecting the ability of various agents to modulate monocyte differentiation into fibrocytes and to detect monocyte defects.

PRIORITY CLAIM

The present application is a continuation-in-part under 35 U.S.C. §120of PCT patent application serial number PCT/US2003/041183, filed Dec.22, 2003 and titled “Methods of Detecting the Inhibition of FibrocyteFormation and Methods and Compositions for Enhancing FibrocyteFormation”, published in English as WO 04/059318 on Jul. 22, 2004; whichclaims priority to the following: U.S. Provisional Patent Applications:U.S. 60/436,046, filed Dec. 23, 2002; U.S. 60/436,027, filed Dec. 23,2002; U.S. 60/515,776, filed Oct. 30, 2003; U.S. 60/519,467, filed Nov.12, 2003; and U.S. 60/525,175 filed Nov. 26, 2003. Pertinent parts ofall above applications are incorporated by reference herein.

TECHNICAL FIELD

The present invention relates to the ability of SAP to suppress thedifferentiation of monocytes into fibrocytes. Accordingly, it mayinclude compositions and methods for increasing such differentiation.These compositions and methods may be useful in a variety ofapplications in which increased fibrocyte formation is beneficial, suchas wound healing. The invention may additionally include methods fordetecting problems in the ability of monocytes to differentiate intofibrocytes or for SAP to inhibit this differentiation. These problemsmay be correlated with a disease or may be drug-induced.

BACKGROUND

Fibrocytes

Inflammation is the coordinated response to tissue injury or infection.The initiating events are mediated by local release of chemotacticfactors, platelet activation, and initiation of the coagulation andcomplement pathways. These events stimulate the local endothelium,promoting the extravasation of neutrophils and monocytes. The secondphase of inflammation is characterized by the influx into the tissue ofcells of the adaptive immune system, including lymphocytes. Thesubsequent resolution phase, when apoptosis of the excess leukocytes andengulfment by tissue macrophages takes place, is also characterized byrepair of tissue damage by stromal cells, such as fibroblasts.

Both IL-4 and IL-13 are potent activators of the fibrotic response. IL-4in known to enhance wound repair and healing. IL-13 has a high degree ofhomology with IL-4 and in many systems they act in a similar manner.However, key differences have been found in the function of these twoproteins in various circumstances. For instance, IL-13 is more dominantin resisting infection by intestinal nematodes and intracellularparasites, such as Leishmania. IL-13 also plays a much more significantrole than IL-4 in asthma. In contrast, IL-4 is more dominant than IL-13in stimulating B cell production of immunoglobulin and in T cellsurvival and differentiation.

TGFβ, which is also known to play a role in wound healing, had beenshown to facilitate fibrocyte differentiation into myofibroblasts, whichare further associated with wound healing.

Although IL-4, IL-13, TGFβ and various other factors are known play arole in the fibrotic response, the source of fibroblasts responsible forrepair of wound lesions or in other fibrotic responses is controversial.The conventional hypothesis suggests that local quiescent fibroblastsmigrate into the affected area, produce extracellular matrix proteins,and promote wound contraction or fibrosis. An alternative hypothesis isthat circulating fibroblast precursors (called fibrocytes) presentwithin the blood migrate to the sites of injury or fibrosis, where theydifferentiate and mediate tissue repair and other fibrotic responses.

Fibrocytes are known to differentiate from a CD14+ peripheral bloodmonocyte precursor population. Fibrocytes express markers of bothhematopoietic cells (CD45, MHC class II, CD34) and stromal cells(collagen types I and III and fibronectin). Mature fibrocytes rapidlyenter sites of tissue injury where they secrete inflammatory cytokines.Once there, fibrocytes can function as antigen presenting cells (APCs),thereby inducing antigen-specific immunity. Fibrocytes are also capableof secreting extracellular matrix proteins, cytokines and pro-angiogenicmolecules, which may aid in wound repair.

Fibrocytes are also associated with a variety of other processes anddisorders. They are associated with the formation of fibrotic lesionsafter Schistosoma japonicum infection in mice and are also implicated infibrosis associated with autoimmune diseases. Fibrocytes have also beenimplicated in pathogenic fibrosis such as that associated with radiationdamage, Lyme disease and pulmonary fibrosis. CD34+ fibrocytes have alsobeen associated with stromal remodeling in pancreatitis and stromalfibrosis, whereas lack of such fibrocytes is associated with pancreatictumors and adenocarcinomas. This correlation may relate to the abilityof fibrocytes to function as APCs. Finally, fibrocytes have been shownto promote angiogenesis by acting on endothelial cells.

Serum Amyloid P

Serum amyloid P (SAP), a member of the pentraxin family of proteins thatinclude C-reactive protein (CRP), is secreted by the liver andcirculates in the blood as stable pentamers. The exact biological roleof SAP is still unclear, although it appears to play a role in both theinitiation and resolution phases of the immune response. SAP binds tosugar residues on the surface of bacteria leading to their opsonisationand engulfment. SAP also binds to free DNA and chromatin generated byapoptotic cells at the resolution of an immune response, thus preventinga secondary inflammatory response. Molecules bound by SAP are removedfrom extracellular areas due to the ability of SAP to bind to all threeclassical Fcγ receptors (FcγR), with a preference for FcγRI (CD64) andFcγRII (CD32). After receptor binding, SAP and any attached molecule arelikely engulfed by the cell.

FcγR are necessary for the binding of IgG to a wide variety ofhematopoietic cells. Peripheral blood monocytes express both CD64 andCD32, whereas tissue macrophages express all three classical FcγR. Asubpopulation of monocytes also express CD16 (FcγRII).

Clustering of FcγR on monocytes by IgG, either bound to pathogens or aspart of an immune complex, initiates a wide variety of biochemicalevents. The initial events following receptor aggregation include theactivation of a series of src kinase proteins. In monocytes, theseinclude lyn, hck and fgr, which phosphorylate tyrosine residues on theITAM motif of the FcR-γ chain associated with FcγRI and FcγRIII, or theITAM motif with the cytoplasmic domain of FcγRII. Phosphorylated ITAMslead to the binding of a second set of src kinases, including syk. Sykhas been shown to be vital for phagocytosis of IgG-coated particles.However, the wide distribution of syk in non-hematopoietic cells and theevidence that syk is involved in both integrin and G-protein coupledreceptor signaling, indicates that this molecule has many functions.

Both SAP and CRP augment phagocytosis and bind to Fcγ receptors on avariety of cells. CRP binds with a high affinity to FcγRII (CD32), alower affinity to FcγRI (CD64), but does not bind FcγRIII (CD16). SAPbinds to all three classical Fcγreceptors, with a preference for FcγRIand FcγRII, particularly FCγRI. Although there are conflictingobservations on the binding of CRP to FcγR, both SAP and CRP have beenshown to bind to Fc receptors and initiate intracellular signalingevents consistent with FcγR ligation.

In human blood serum, males normally have approximately 32 μg/ml+/−7μg/ml of SAP, with a range of 12-50 μg/ml being normal. Human femalesgenerally have approximately 24 μg/ml+/−8 μg/ml of SAP in blood serum,with a range of 8-55 μg/ml being normal. In human cerebral spinal fluidthere is normally approximately 12.8 ng/ml SAP in human males andapproximately 8.5 ng/ml in females. Combining male and female data, thenormal SAP level in human serum is 26 μg/ml+/−8 μg/ml with a range of12-55 μg/ml being normal. (The above serum levels are expressed as mean+/−standard deviation.)

SAP has been investigated primarily in relation to its role inamyloidosis. Recently, a drug,R-1-[6-[R-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC) was developed to deplete SAP andthereby treat amyloidosis. However, this drug appears to have beenapplied systemically and not to have been used to treat wound healing orto have other localized or systemic effects.

Agar has been previously used as a wound dressing. However, it is notclear whether such previous wound dressings were capable of depletingSAP because they may not have contained appropriate chemical moieties ormay have been used inappropriately. In any event, these previous wounddressing do not appear to have incorporated any additional wound healingfactors. Further the dressings appear to have been used only forexternal wounds. Finally, it does not appear that these dressingsincorporated purified SAP depleting chemicals or enhanced levelsthereof.

SUMMARY

The present invention may include compositions and methods for bindingSAP. Compositions operable to bind SAP may include CPHPC, the4,6-pyruvate acetyl of beta-D-galactopyranose, phosphoethanolamines, andanti-SAP antibodies or fragments thereof. Such binding may occur invivo.

The invention may also include compositions and methods for thedepletion of SAP levels in a sample. The sample may be located in vitroor in vivo. In vivo the sample may include an entire organism or aportion thereof and depletion may be systemic or may be confined to aparticular area, such as an organ or wound. The compositions may includethose supplied directly or produced in the sample, for instance throughexpression of a transgene. Compositions operable to deplete SAP mayinclude CPHPC, high EEO agarose, the 4,6-pyruvate acetyl ofbeta-D-galactopyranose, phosphoethanolamine, and anti-SAP antibodies orfragments thereof. SAP levels in a sample may also be depleted byinterfering with its initial production or increasing degradation.

The invention may also include compositions and methods for thesuppression of SAP activity. Suppression may be in a sample and mayoccur in vitro or in vivo. Compositions also include compositionssupplied directly to a sample and those produced in the sample, such asby expression of a transgene. These compositions may act by decreasingSAP formation, decreasing the ability of SAP proteins to interact withmonocytes, decreasing the ability of SAP proteins to interact withcofactors or decreasing the level of such cofactors, and interferingwith SAP-induced signaling in monocytes, such as a pathway triggered bySAP binding to an FcγR. Compositions operable to suppress SAP activitymay include anti-SAP antibodies and fragments thereof, particularlythose targeted the Fc-binding region.

The invention may additionally include methods and compositions forpromoting wound healing by depleting or, suppressing SAP in the regionof a wound. Compositions may also include additional wound healingfactors. In specific embodiments of the invention, wound healingcompositions may include high EEO agarose, phosphoethanolamine agaroseor Ca²⁺ and combinations thereof. Cytokines such as IL-13, IL-4 and TGFβmay be added to these compositions.

Yet another aspect of the invention relates to compositions and methodsfor promoting fibrocyte formation by providing IL-4, IL-13 or acombination of the two to monocytes. The monocytes may be located invitro or in vivo. IL-4 and IL-13 may be provided by an extraneoussource, or endogenous production may be increased.

Finally, the invention may include assays to detect the ability of asample to modulate fibrocyte differentiation from monocytes. In oneembodiment, normal monocytes may be supplied with the sample. The samplemay include normal SAP. It may also include SAP or a biological fluidfrom a patient such as a patient with a wound healing disorder, or itmay include a potential drug. In another embodiment, the sample mayinclude normal SAP while the monocytes may be derived from a patient andmay be abnormal. In either type of assay, the effects on monocytedifferentiation into fibrocytes may be compared with a normal control todetect any increases or decreases in monocyte differentiation ascompared to normal.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed descriptionpresented herein.

FIGS. 1A-1B illustrate the effects of serum and plasma on the rapiddifferentiation of fibroblast-like cells. In FIG. 1A peripheral bloodmononuclear cells (PBMC) at 2.5×10⁵ per ml were cultured in serum-freemedium for 3 or 6 days in the presence or absence of 0.1% human serumand then examined by microscopy for the appearance of fibroblast-likecells. Bar is 100 μm.

In FIG. 1B PBMC at 2.5×10⁵ per ml were cultured in serum-free medium for6 days in dilutions of human plasma. Cells were then air-dried, fixed,stained, and fibrocytes were enumerated by morphology. Results areexpressed as mean±SD of the number of fibrocytes per 2.5×10⁵ PBMCs (n=5experiments). Stars indicate values that are statistically significantdifferences from the 0 SAP value.

FIG. 2 illustrates the expression of surface molecules onfibroblast-like cells. PBMC were cultured on glass slides in serum-freemedium for 6 days. Cells were air-dried and analyzed byimmunohistochemistry. Monoclonal antibodies used were as indicated, andidentified by biotin-conjugated goat anti-mouse Ig followed byExtrAvidin peroxidase. Cells were counterstained with Mayer'shaematoxylin to identify nuclei. Positive staining was identified bybrown staining, nuclei were counterstained blue. An insert for CD83 wasused to indicate positive staining on a dendritic cell.

FIG. 3 illustrates the characterization of the molecule present inplasma that inhibits fibrocyte differentiation. Citrated plasma wastreated with BaCl₂ and the precipitated material was collected bycentrifugation and dialyzed against 10 mM sodium phosphate containing 10mM EDTA and protease inhibitors. This material was then fractionated byheparin and ion exchange chromatography.

In FIG. 3A fractions were analyzed by PAGE on a 4-20% reducing gel andstained with coomassie blue. M indicates molecular weight markers. Lane1 contained plasma, lane 2 contained BaCl₂ supernatant, lane 3 containedwash 1, lane 4 contained wash 2, lane 5 contained BaCl₂ precipitate,lane 6 contained BaCl₂ precipitate, lane 7 contained heparin flowthrough, lane 8 contained the heparin fraction, lane 9 contained High Qflow through, lane contained the 10 High Q fraction, lane 11 containedthe gel purified fraction. Lanes 1-5 diluted 1:500 in sodium phosphatebuffer, lanes 6-11 undiluted.

Active fractions eluted off the High Q ion exchange column and gelslices were analyzed by 4-20% PAGE on a native gel in FIG. 3B and areducing gel in FIG. 3C. NM indicates native gel markers, RM indicatesreduced gel markers, in FIG. 3B lanes 1-3 are control gel samples, lane4 contained active fraction. In FIG. 3D fractions were assessed bywestern blotting, using a rabbit anti-SAP antibody. Lanes 1-11correspond to those in FIG. 3A.

FIG. 4 shows the inhibition of fibrocyte formation by SAP, but not CRPor other plasma proteins. PBMC at 2.5×10⁵ per ml were cultured inserum-free medium for 6 days in the presence of commercially availablepurified SAP (filled square), CRP (open square), Protein S (opendiamond) or C4b (open circle) and then examined for the appearance offibroblast-like cells. Cells were then air-dried, fixed, stained andfibrocytes enumerated by morphology. Results are mean±SD of fibrocytesper 2.5×10⁵ PBMC (n=3 separate experiments).

FIGS. 5A-5B show the effect of depletion of SAP from plasma in afibrocyte differentiation assay.

FIG. 5A shows the effect on fibrocyte differentiation of depleting SAPfrom plasma with BioGel agarose beads. Number of fibrocytes found in anassay supplied with either plasma (open square) or BioGel depletedplasma (filled square) at a variety of dilutions is shown.

FIG. 5B shows the number of fibrocytes formed in an assay performed withno plasma or equal dilutions of plasma, BioGel SAP depleted plasma, oranti-SAP antibody depleted plasma. Stars indicate statisticallysignificant differences.

FIG. 6 shows initial skin incisions on three different rats to betreated with saline, saline with CaCl₂, or agarose with saline andCaCl₂.

FIGS. 7A-7B show healing of the skin incisions shown in FIG. 6. FIG. 7Ashows healing of skin incisions on three different rats after one day oftreatment with either saline, saline with CaCl₂, or agarose with salineand CaCl₂. FIG. 7B shows a comparison of initial skin incisions on threedifferent rats and healing after one day of treatment with eithersaline, saline with CaCl₂, or agarose with saline and CaCl₂.

FIGS. 8A-8B also show healing of skin incisions on rats. FIG. 8A showshealing of skin incisions on three different rats after two days oftreatment with either saline, saline with CaCl₂, or agarose with salineand CaCl₂. FIG. 8B shows a comparison of initial skin incisions on threedifferent rats and healing after one and two days of treatment witheither saline, saline with CaCl₂, or agarose with saline and CaCl₂.

FIG. 9 shows the effects of various cytokines on promotion of fibrocytedifferentiation.

FIG. 10 shows an experimental setup in a porcine model.

FIG. 11 shows the steps of an epidermal migration assessment in aporcine model. A: wound excision; B: placement of specimen in sodiumbromide for incubation; C: placement of specimen on glass slide forseparation; D: separation of specimen; D: placement of epidermalspecimen on cardboard for permanent record.

FIG. 12 shows the combined healing data from porcine wound healingstudies. Day after wounding is indicated on the x axis.

DETAILED DESCRIPTION

Fibrocytes are a distinct population of fibroblast-like cells derivedfrom peripheral blood monocytes. Culturing CD14+ peripheral bloodmonocytes in the absence of serum or plasma leads to the rapiddifferentiation of fibrocytes. This process normally occurs within 48-72hours and is suppressed by the presence of blood serum or plasma.Experiments described further herein have determined that thissuppression is caused by SAP. Additional experiments have determinedthat, when monocytes are cultured in serum-free medium, differentiationinto fibrocytes is enhanced by the presence of IL-4 or IL-13.

Binding of SAP

The present invention may include compositions and methods for bindingSAP. Compositions may include CPHPC, the 4,6-pyruvate acetyl ofbeta-D-galactopyranose, ethanolamines, anti-SAP antibodies or fragmentsthereof, and DNA. Agarose may also be used to bind SAP. For example,High EEO agarose (Fisher Scientific Internation Inc., NH), Low EEOagarose (Fisher Scientific Internation Inc., NH), SeaKem® ME agarose(Cambrex Bioscience, NJ), SeaKem® SP agarose (Cambrex Bioscience, NJ),Bio-Gel A (BioRad Laboratories, CA), SP-Sepharose (Amersham Biosciences,UK) CL-Sepharose (Amersham Biosciences, UK), Heparin-agarose, Asparticacid-agarose and Poly-lysine-agarose and derivatized agarose may all beused in embodiments of the invention.

These compositions may include purified chemicals, or the chemicals maybe attached to another compound, for example a much larger compound,such as agarose or a biocompatible polymer (e.g. PEG, poly(amino acids)such as poly(glutamic acid), chitosan, other polysaccharides, and otherbiological polymers, or chemically modified versions thereof). Bindingmay occur in vitro or in vivo.

In a specific embodiment, SAP may be bound by a composition includingapproximately 1% w/v high EEO agarose. The composition may also includea cation, such as Mg²⁺ or Ca²⁺. For example, the agarose may includeapproximately 5 mM CaCl₂.

In other embodiments, the composition may include an antibody orantibody fragment that targets the portion of SAP functional ininhibiting fibrocyte formation from monocytes. In an exemplaryembodiment, the functional portion of SAP may be selected from theregion that does not share sequence homology with CRP, which has noeffect on fibrocyte formation. For instance amino acids 65-89KERVGEYSLYIGRHKVTSKVIEKFP (SEQ ID NO:1) of SAP are not homologous toCRP. Amino acids 170-181 ILSAYQGTPLPA (SEQ ID NO:2) and 192-205IRGYVIIKPLV (SEQ ID NO:3) are also not homologous. Additionally a numberof single amino acid differences between the two proteins are known andmay result in functional differences.

Depletion of SAP

Other aspects of the invention relate to compositions and methods forthe depletion of SAP levels in a sample. The sample may be located invitro or in vivo. In vitro samples may include tissue cultures,bioreactors, tissue engineering scaffolds and biopsies. In vivo thesample may include an entire organism or a portion thereof such as anorgan or injury site. Depletion in vivo may be systemic or it may beconfined to a particular area, such as an organ or wound.

Compositions for depletion of SAP may include those supplied directly tothe sample. For instance all of the binding agents mentioned above maybe supplied directly to the sample. They may be supplied in any form orformulation although those that do not substantially interfere withdesired outcomes for the sample may be preferred.

Compositions for the depletion of SAP may also be produced in thesample, or in an organism containing the sample. For example, atransgene encoding an anti-SAP antibody may be introduced into thesample.

SAP may be directly depleted by a material that binds or sequesters SAP,such as agarose, CPHPC, 4,6-pyruvate acetyl of beta-D-galactopyranose,phosphoethanolamine agarose, anti-SAP antibodies, DNA analogs andcarbohydrate analogs.

Depletion may also occur by degradation or inactivation of SAP such asthrough the use of SAP-specific proteases.

Other compositions may increase the rate of uptake of SAP and thisdecrease its availability.

Finally, SAP levels may also be depleted by interfering with its initialproduction or increasing its degradation. In a specific embodiment, SAPlevels may be depleted in vivo by administering a composition thatinhibits SAP production. Because SAP is primarily produced in the liver,in vivo suppression of SAP production should be easily attained, butwill be systemic. Compositions that interfere with SAP production mayact upon a signaling pathway that modulates SAP production.

Suppression of SAP Activity

The invention may also include compositions and methods for thesuppression of SAP activity. Suppression may be in a sample and mayoccur in vitro or in vivo. Compositions may also include compositionssupplied directly to a sample and those produced in the sample. Manysuch compositions may be SAP-binding compositions described above. Inparticular, compositions for the suppression of SAP activity may includeantibodies selected as described above to bind to specific regions ofSAP not homologous to CRP. Antibodies may also target the region of SAPthat binds to FcγR or may compete with SAP for binding to the thesereceptors. Small peptides may also be able to block SAP binding to theFcγR or compete with SAP for binding to these receptors.

Compositions that suppress SAP activity may act by a variety ofmechanisms including but not limited to: decreasing the ability of SAPproteins to interact with monocytes, decreasing the ability of SAPproteins to interact with cofactors or decreasing the level of suchcofactors, and interfering with SAP-induced signaling in monocytes, suchas a pathway triggered by SAP binding to an FcγR. This pathway isdescribed in detail in Daeron, Marc, “Fc Receptor Biology”, Annu. Rev.Immunology 15:203-34 (1997). In an exemplary embodiment a portion of thepathway that is not shared with other signaling cascades or only alimited number of non-critical signaling cascades may be selected forinterference to minimize side-effects. For example, a composition mayinterfere with the Fc pathway by blocking syk kinase.

Effects of IL-4 and IL-13

Yet another aspect of the invention relates to compositions and methodsfor promoting fibrocyte formation by providing IL-4, IL-13 or acombination of the two to monocytes. The monocytes may be located invitro or in vivo. IL-4 and IL-13 may be provided by an extraneoussource, or endogenous production may be increased. More specifically,IL-4 or IL-13 may be provided at concentrations of between approximately0.1 and 10 ng/ml.

Uses for Modulating Fibrocyte Formation

Depletion or suppression of SAP or supply of IL-4 or IL-13 in a samplemay be used to increase fibrocyte differentiation from monocytes. Thiseffect has many uses both in vitro and in vivo. For example, in vitroincreased fibrocyte formation may be useful in tissue engineering.Production of fibrocytes in areas requiring vascularization may induceangiogenesis. In vitro, increased differentiation of monocytes to formfibrocytes may also be used for internal tissue engineering or forinducing angiogenesis in areas in need of new vasculature.

Additionally, increasing differentiation of monocytes into fibrocytes invivo may promote wound healing or may be used for cosmetic surgeryapplications. Wound healing may benefit, inter alia, from the ability offibrocytes to further differentiate into other cells such asmyofibroblasts and from angiogenic effects of fibrocytes as well as thefrom their ability to function as APCs, thereby assisting in preventionor control of infection.

Because of the ability of fibrocytes to function as APCs, areas ofchronic infection or areas that are infected but not readily reached bythe immune system, such as cartilage, may also benefit from increasedmonocyte differentiation into fibrocytes.

Because pancreatic tumors and adenocarcinomas show lower levels offibrocytes, increasing differentiation of monocytes into fibrocytes inthese tissues may help slow the tumor progression or aid in remission.

Specific Formulations

Some compositions of the present invention may be provided in a varietyof formulations.

In a specific example, the invention may include methods andcompositions for promoting wound healing by depleting or suppressing SAPin the region of a wound. These wound healing compositions may includeCPHPC, anti-SAP antibodies, 4,6-pyruvate acetyl of B-D-galactopyranose,such as found on high EEO agarose, ethanolamines, such as those found onphosphoethanolamine agarose, Ca²⁺, and combinations thereof. Cytokinessuch as IL-13, IL-4, FGF and TGFβ may be added to these compositions.

In many patients only localized SAP depletion or inhibition orinterference with a SAP-modulated pathway may be desirable. Manycompositions within the scope of the present invention may beadministered locally to such patients. For instance, administration of acomposition may be topical, such as in an ointment, cream, solid, spray,vapor or wound dressing. Such topical formulations may include alcohol,water, disinfectants, other volatile substances, or any otherpharmaceutically active agents, such as antibiotics and anti-infectiveagents, or pharmaceutically acceptable carriers. Local administrationmay also be by localized injection of a composition alone or incombination with another pharmaceutically active agent orpharmaceutically acceptable carrier.

Patients for whom localized administration of compositions that increasemonocyte differentiation into fibrocytes may be advisable include butare not limited to: mild to moderate burn patients; patients who havesuffered lacerations, including those inflicted during surgicalprocedures; patients suffering from diabetic complications, such asulcers; patients with pressure ulcers or areas of low circulationinternally; patients with abrasions, minor contusions or puncturewounds; patients with bullet or shrapnel wounds; patients with openfractures; patients in need of tissue growth for tissue engineering orcosmetic reasons; and immunosuppressed, hemophiliac or other patientswho are likely to benefit from the more rapid healing of most wounds.

For patients with severe or numerous wounds or other disorders, moregeneral administration of a composition to promote fibrocyte formationthrough an IV or other systemic injection may be appropriate. Patientsfor whom systemic administration of a SAP depleting or inhibiting agentmay be helpful include, but are not limited to: severe burn patients;later stage peripheral arterial occlusive disease patients; and patientswith general wound healing disorders.

Some formulations may be appropriate for local or systemicadministration. Additionally, the therapeutic agent may be supplied in asolid form, such as a powder, then reconstituted to produce theformulation ultimately administered to a patient.

In an exemplary embodiment for the treatment of wound healing, high EEOagarose or phosphoethanolamine agarose may be administered as a woulddressing. In this embodiment, the agarose may be at a concentration ofapproximately 1% (w/v) and may also contain approximately 5 mM CaCl₂.The wound dressing may be applied for any period of time. Although itmay be applied continuously until the wound has closed (approximatelytwo days or more), it may also only be applied for a short initialperiod, such as 12 hours. This initial removal of SAP from the wound maybe sufficient to induce increased differentiation of monocytes intofibrocytes and improve wound healing.

In another exemplary embodiment, CPHPC may be administered systemicallyto promote healing of widespread or recalcitrant wounds. CPHPC has beenpreviously administered in a range of 1.5 to 15 mg/kg/day by osmoticpumps in mice in amyloidosis experiments. CPHPC has also beenadministered in 1 mg/ml water concentrations in drinking water for mice.A 20 g mouse drinks approximately 3 ml of water per day, resulting in anintake of approximately 0.15 CPHPC mg/kg/day. Such ranges are thereforelikely safe in humans to reduce SAP levels, although different rangesmay provide optimal benefit for wound healing.

In other embodiments, the compositions may be provided in or onprosthetic devices, particularly surgically implanted prostheticdevices.

In some embodiments, the compositions may be provided in a slow-releasegel or dressing, such as a plastic substrate. Compositions may also beprovided as hydrogels.

Monocyte Differentiation Assays

Another aspect of the invention relates to assays to detect the abilityof a sample to modulate fibrocyte differentiation from monocytes. Inserum-free medium, normal monocytes form fibrocytes in two to threedays. Normal serum, blood or other biological fluids suppress theformation of fibrocytes from normal monocytes over a specific dilutionrange. Thus the assay may be used to test whether a sample can modulatedifferentiation of monocytes into fibrocytes in serum-free medium. Itmay also be used to determine whether sample monocytes differentiatenormally into fibrocytes in serum-free medium and if they respondnormally to serum, SAP or other factors affecting this differentiation.

In a specific embodiment, the assay may be used to determine whether apatient's biological fluid has a decreased or increased ability tosuppress monocyte differentiation into fibrocytes. If suppression by SAPis to be tested, any biological fluid in which SAP is normally ortransiently present may be used, including whole blood, serum, plasma,synovial fluid, cerebral spinal fluid and bronchial fluid. An increasedability to suppress monocyte differentiation may be indicative of awound healing disorder or other disorders, or the propensity to developsuch a disorder. Although in many patients an increased ability of abiological fluid to suppress fibrocyte formation may be due to lowlevels of SAP, this is not necessarily the case. SAP may be present atnormal levels, but exhibit decreased suppressive activity due to defectsin the SAP itself or the absence or presence of a cofactor or othermolecule. Methods of determining the more precise nature of thesuppression problem, such as use of ELISAs, electrophoresis, andfractionation will be apparent to one skilled in the art.

The methodology described above may also be used to determine whethercertain potential drugs that affect fibrocyte differentiation may or maynot be appropriate for a patient.

In another specific embodiment, the assay may be used to determine if apatient's monocytes are able to differentiate into fibrocytes inserum-free medium and if they respond normally to a biological fluid,SAP or another composition. More particularly, if a patient with woundhealing problems appears to have normal levels of SAP, it may beadvisable to obtain a sample of the patient's monocytes to determine ifthey are able to differentiate in the absence of serum or SAP.

Finally, in another specific example, the assay may be used to test theeffects of a drug or other composition on monocyte differentiation intofibrocytes. The assay may be used in this manner to identify potentialdrugs designed to modulate fibrocyte formation, or it may be used toscreen for any potential adverse effects of drugs intended for otheruses.

The following examples are included to demonstrate specific embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

EXAMPLES Example 1 Inhibition of Fibrocyte Formation

While examining the possible role of cell density in the survival ofperipheral blood T cells, it was observed that in serum-free medium PBMCgave rise to a population of fibroblast-like cells. These cells wereadherent and had a spindle-shaped morphology (FIG. 1A). Approximately0.5-1% of PBMC differentiated into fibroblast-like cells in serum-freemedium, and this occurred in tissue culture treated plasticware andborosilicate and standard glass slides.

The rapid appearance of these cells, within 3 days of culture, wasinhibited by human serum or plasma. To examine this process in moredetail, PBMC were cultured at 5×10⁵ cells per ml in serum-free mediumcontaining increasing concentrations of human plasma for 6 days. Whenplasma was present at concentrations between 10% and 0.5%, thefibroblast-like cells did not differentiate (FIG. 1B). However, at orbelow 0.1% serum, fibroblast-like cells rapidly developed. The activityin the serum that inhibited fibrocyte formation was retained by a 30 kDacutoff spin-filter (data not shown). If serum was heated to 56° C. for30 minutes, the efficacy was reduced 10 fold, and heating to 95° C.abolished the inhibitory activity (data not shown).

These data suggested that that the inhibitory factor is a protein. Asthe inhibitory factor was present in human serum, it indicated that theactivity was unlikely to be involved with the coagulation system. Theinhibitory factor also appeared to be an evolutionary conserved proteinas bovine, equine, caprine, and rat sera were also able to inhibit theappearance of these fibroblast-like cells (data not shown).

Example 2 Characterization of Fibroblast-Like Cells

The differentiation of these fibroblast-like cells from peripheral bloodsuggested that they might be peripheral blood fibrocytes. Fibrocytes area population derived from peripheral blood monocytes that differentiatein vitro and in vivo into fibroblast-like cells. They rapidly enterwound sites and are capable of presenting antigens to T cells. Theirphenotype is composed of both haematopoietic markers, such as CD45 andMHC class II, and stromal markers, such as collagen I and fibronectin.However in order to identify these cells, PBMC were generally culturedfor 1-2 weeks in medium containing serum.

To characterize whether the cells observed in the system werefibrocytes, PBMC were depleted of T cells with anti-CD3, B cells withanti-CD19, monocytes with anti-CD14 or all antigen presenting cells withanti-HLA class II and then cultured in serum-free conditions for 6 days.Depletion of PBMC with anti-CD3 or anti-CD19 did not depletefibroblast-like cells from PBMC when cultured in serum-free cultures(data not shown). Depletion of antigen presenting cells with anti-HLAclass II or monocytes with anti-CD14 antibody did prevent the appearanceof fibroblast-like cells, indicating that the fibroblast-like cells arederived from monocytes and not a dendritic cell population.

To further characterize the fibroblast-like cells, PBMC were cultured inserum-free medium for 5 days on glass slides. Cells were then air-dried,fixed in acetone and labeled with a variety of antibodies (Table 1 andFIG. 2). Fibrocytes express CD11a, CD11b, CD45, CD80, CD86, MHC classII, collagen I, fibronectin, the chemokine receptors CCR3, CCR5, CCR7,CXCR4 and α-smooth muscle actin. In the above culture conditions, thefibroblast-like cells in the present experiment also expressed all thesemarkers. Fibrocytes are negative for CD1a, CD3, CD19, CD38 and vWF, aswere the fibroblast-like cells in the present experiment. Based on thesedata it appears that the fibroblast-like cells observed in theexperiments were fibrocytes. Further experiments were conducted toextend this phenotype. In the above conditions, the fibrocytes expressedseveral β1 integrins including α1 (CD49a), α2 (CD49b), α5 (CD49e), β1(CD29) and β3 (CD61) along with high levels of β2 (CD18), but werenegative for α3, α4, α6 α4β7, αE and CLA (FIG. 2 and Table 1).

TABLE 1 Expression of surface markers on Fibrocytes Marker AlternativeName Fibrocyte Expression CD11a LFA-1 positive CD11b Mac-1 positiveCD11c positive GD13 positive GD18 β2 integrin positive CD29 β1 integrinpositive CD34 positive CD40 weak positive CD45 LCA positive CD49a α1integrin weak positive CD49b α2 integrin negative CD49e α5 integrinpositive CD51 positive CD54 ICAM-1 positive CD58 LFA-3 positive CD61 β3integrin positive CD80 B7-1 weak positive CD86 B7-2 positive GD105Endoglin positive CD148 positive MHC Class II positive CD162 PSGL-1positive CCR1 weak positive CCR3 weak positive CCR4 weak positive CCR5weak positive CCR7 weak positive CCR9 weak positive CXCR1 positive CXCR3positive CXCR4 weak positive Collagen I positive Collagen III positiveFibronectin positive α Smooth Muscle Actin positive Vimentin positiveCD1a negative CD3 negative CD10 negative CD14 negative GD19 negativeCD25 negative CD27 negative CD28 negative CD38 negative CD49C α3integrin negative CD49d α4 integrin negative CD49f α6 integrin negativeCD69 negative CD70 CD27-L negative CD90 negative CD103 αE integrinnegative GD109 negative CD154 CD40-L negative α4β7 negative CLA negativeCCR2 negative CCR6 negative CXCR2 negative CXCR5 negative CXCR6 negativeCytokeratin negative vWF negative

To obtain the data in Table 1, PBMC were cultured in the wells of 8 wellglass slides at 2.5×10⁵ cells per ml (400 μl per well) in serum-freemedium for 6 days. Cells were then air dried, fixed in acetone andstained by immunoperoxidase. Cells were scored positive or negative forthe indicated antigens, compared to isotype-matched control antibodies.

Example 3 Characterization of the Fibrocyte Inhibitory Factor

The initial characterization of the serum factor that prevents rapidfibrocyte differentiation indicated that the factor was aheparin-binding molecule that eluted off an ion exchange column (High Q)as one of four proteins. By sequencing tryptic fragments of protein in aband cut from a native gel, one of these proteins was identified asC4b-binding protein (C4BP). C4b-binding protein is a 570 kDa protein,composed of seven alpha chains (70 kDa) and usually a single beta chain(40 kDa), which is involved in regulating the decay of C4b and C2acomponents of the complement system. C4BP also interacts with thevitamin K-dependent anticoagulant protein S. The C4BP/Protein S complexcan be purified from serum or plasma using BaCl2 precipitation.

To assess whether C4BP, or an associated protein, was the factorresponsible for inhibiting fibrocyte differentiation, citrated plasmawas treated with BaCl2. The inhibitory factor was present in the BaCl2precipitate (FIG. 3 and Table 2). This fraction was applied to a heparincolumn and the fractions, eluted by increasing concentrations of NaCl,were assessed for their ability to inhibit monocyte to fibrocytedifferentiation in serum free medium. The active factor was eluted offthe heparin column in a peak at 200 mM NaCl (FIG. 3 and Table 2). Aslight increase in the yield suggested that this step may have removed afactor that slightly interfered with the activity of the factor.

The fractions from the 200 mM peak were pooled and further fractionatedby High Q ion exchange chromatography. A small peak eluting at 300 mMNaCl contained activity that inhibited fibrocyte differentiation.Analysis of the proteins present in this fraction indicated that themajor band was a 27 kDa protein. Although the ion exchangechromatography led to a reduction in the amount of SAP recovered (FIG.3A, lanes 8-10 and FIG. 3D, lanes 8-10) this step did remove severalcontaminating proteins. After the ion exchange step the only discernablecontaminant was albumin at 65 kDa (FIG. 3A, lane 10).

The high Q fraction was concentrated and fractionated by electrophoresison a non-denaturing polyacrylamide gel, followed by elution of thematerial in gel slices. A single band that migrated at approximately 140kDa was able to inhibit differentiation (FIG. 3B). This band had amolecular weight of 27 kDa on a reducing polyacrylamide gel, suggestingthat the native conformation of the protein was a pentamer (FIG. 3C).This band was excised from the gel, digested with trypsin and analyzedby MALDI mass spectrometry. Three major and two minor peptides wereidentified: VFVFPR (SEQ ID NO: 4), VGEYSLYIGR (SEQ ID NO: 5),AYSLFSYNTQGR (SEQ ID NO: 6), QGYFVEAQPK (SEQ ID NO: 7) and IVLGQEQDSYGGK(SEQ ID NO: 8). These sequences exactly matched amino acid sequences8-13, 68-77, 46-57, 121-130 and 131-143 of serum amyloid P.

To confirm that the active fractions contained SAP, fractions collectedfrom column chromatography were analyzed by western blotting (FIG. 3D).The presence of SAP at 27 kDa was detected in all fractions thatinhibited fibrocyte differentiation (FIG. 3D, lanes 6, 8, 10 and 11). Aconsiderable amount of SAP was present in the supernatant from the BaCl2precipitation step indicating that this procedure was inefficient, withthe recovery of only approximately 10-15% of the fibrocyte inhibitoryactivity in the BaCl2 pellet (FIG. 3A lane 2). In order to remove theknown problem of anti-SAP antibodies binding to immunoglobulins whenused with western blotting, the antibody was pre-incubated with humanIgG bound to agarose. Fractions were also analyzed for the presence ofCRP, C4BP and protein S. Western blotting indicated that C4BP andProtein S were present in plasma, and in the barium precipitation, butwere absent from the active fractions collected from heparinchromatography (data not shown).

TABLE 2 Recovery of protein and fibrocyte inhibitory activity fromfractionated human plasma Total Volume Protein protein Yield (ml) (mg/ml) (mg) (%) Plasma 250 70 17,500 100 BaCl₂ 240 60 14,400 82.3 supernatantBaCl₂ 31 1 31 0.18 precipitate Heparin 4.3 0.25 1.075 0.006 fractionHigh Q 1.96 0.05 0.098 0.00056 fraction Gel slice 0.075 0.025 0.00180.00001 Total Specific Activity activity Yield activity (U/ml) (U) (%)(U/mg) Plasma 10,000 2.5 × 10⁶ 100 143 BaCl₂ 6,666 1.6 × 10⁶ 64 111supernatant BaCl₂ 1,666 5.1 × 10⁴ 2 1,645 precipitate Heparin 500 2,1500.086 2000 fraction High Q 400 720 0.029 7,300 fraction Gel slice 2000150 0.006 80,000

Plasma was fractionated by BaCl₂ precipitation, heparin and ion exchangechromatography. Protein concentrations were assessed byspectrophotometry at 280 nm. Inhibition of fibrocyte differentiation wasassessed by morphology. The fibrocyte inhibitory activity of a samplewas defined as the reciprocal of the dilution at which it inhibitedfibrocyte differentiation by 50%, when added to serum-free medium.

SAP may also be detected by ELISA using the following methodology:

Maxisorb 96 well plates (Nalge Nunc International, Rochester, N.Y.) werecoated overnight at 4° C. with monoclonal anti-SAP antibody (SAP-5,Sigma) in 50 mM sodium carbonate buffer pH 9.5. Plates were thenincubated in Tris buffered saline pH 7.4 (TBS) containing 4% BSA (TBS-4%BSA) to inhibit non-specific binding. Serum and purified proteins werediluted to 1/1000 in TBS-4% BSA, to prevent SAP from aggregating andincubated for 60 minutes at 37° C. Plates were then washed in TBScontaining 0.05% Tween-20. Polyclonal rabbit anti-SAP antibody(BioGenesis) diluted 1/5000 in TBS-4% BSA was used as the detectingantibody. After washing, 100 pg/ml biotinylated goat F(ab)₂ anti-rabbit(Southern Biotechnology Inc.) diluted in TBS-4% BSA was added for 60minutes. Biotinylated antibodies were detected by ExtrAvidin peroxidase(Sigma). Undiluted peroxidase substrate 3,3,5,5-tetramethylbenzidine(TMB, Sigma) was incubated for 5 minutes at room temperature before thereaction was stopped by 1N HCl and read at 450 nm (BioTek Instruments,Winooska, Vt.). The assay was sensitive to 200 pg/ml.

Example 4 Specificity of Serum Amyloid P

Serum amyloid P is a constitutive plasma protein and is closely relatedto CRP, the major acute phase protein in humans. To assess whether otherplasma proteins could also inhibit the differentiation of fibrocytes,PBMC were cultured in serum-free medium in the presence of commerciallyavailable purified SAP, CRP, C4b or Protein S. The commerciallyavailable SAP was purified using calcium-dependent affinitychromatography on unsubstituted agarose. Of the proteins tested, onlySAP was able to inhibit fibrocyte differentiation, with maximalinhibitory activity at 1 μg/ml (FIG. 4). A dilution curve indicated thatthe commercially available SAP has approximately 6.6×10³ units/mg ofactivity (FIG. 4). Serum and plasma contain between 30-50 μg/ml SAP.Fibrocytes began to appear at a plasma dilution of 0.5%, which would beapproximately 0.15-0.25 μg/ml SAP, which is comparable to the thresholdconcentration of purified SAP. The data showing that SAP purified usingtwo different procedures inhibits fibrocyte differentiation stronglysuggests that SAP inhibits fibrocyte differentiation.

Although these data indicate that SAP is capable of inhibiting fibrocytedevelopment and SAP purifies in a manner that indicates that it is theactive factor in plasma, it was not determined whether depletion of SAPfrom plasma and serum would negate the inhibition. Accordingly, SAP wasdepleted from plasma using agarose beads (BioGel A, BioRad). Plasma wasdiluted to 20% in 100 mM Tris pH 8, 150 mM NaCl, 5 mM CaCl₂ buffer andmixed with 1 ml agarose beads for 2 hours at 4° C. Beads were thenremoved by centrifugation and the process repeated. This depleted plasmawas then assessed for its ability to inhibit fibrocyte differentiation.The control plasma diluted to 20% in 100 mM Tris pH 8, 150 mM NaCl, 5 mMCaCl₂ buffer had a similar dilution curve to that observed withuntreated plasma. In contrast, the bead-treated plasma was less able toinhibit fibrocyte differentiation at intermediate levels of plasma.These data, along with the ability of purified SAP to inhibit fibrocytedifferentiation, strongly suggest that SAP is the active factor in serumand plasma that inhibits fibrocyte differentiation. (See FIG. 5A).

Plasma was also depleted of SAP using protein G beads coated withanti-SAP antibodies. Removal of SAP led to a significant reduction inthe ability of plasma to inhibit fibrocyte differentiation compared withplasma, or plasma treated with beads coated with control antibodies(p<0.05) (FIG. 5B). The beads coated with control antibodies did removesome of the fibrocyte-inhibitory activity from plasma, but this was notsignificantly different from cells cultured with plasma. This probablyreflects SAP binding to the agarose in the protein G beads. These data,together with the ability of purified SAP to inhibit fibrocytedifferentiation, strongly suggest that SAP is the active factor in serumand plasma that inhibits fibrocyte differentiation.

Example 5 Antibodies and Proteins

Purified human CRP, serum amyloid P, protein S and C4b were purchasedfrom Calbiochem (San Diego, Calif.). Monoclonal antibodies to CD1a, CD3,CD11a, CD11b, CD11c, CD14, CD16, CD19, CD34, CD40, Pan CD45, CD64, CD83,CD90, HLA-DR/DP/DQ, mouse IgM, mouse IgG1 and mouse IgG2a were from BDPharmingen (BD Biosciences, San Diego, Calif.). Chemokine receptorantibodies were purchased from R and D Systems (Minneapolis, Minn.).Rabbit anti-collagen I was from Chemicon International (Temecula,Calif.), monoclonal C4b-binding protein was from Green MountainAntibodies (Burlington, Vt.), sheep anti human C4b-binding protein wasfrom The Binding Site (Birmingham, UK), monoclonal anti-CRP was fromSigma (St. Louis, Mo.). Polyclonal rabbit anti-protein S was fromBiogenesis (Poole, Dorset, UK).

Example 6 Cell Separation

Peripheral blood mononuclear cells were isolated from buffy coats (GulfCoast Regional Blood Center, Houston, Tex.) by Ficoll-Paque (AmershamBiosciences, Piscataway, N.J., USA) centrifugation for 40 minutes at400×g. Depletion of specified leukocyte subsets was performed usingnegative selection using magnetic Dynabeads (Dynal Biotech Inc., LakeSuccess, N.Y.), as described previously. Briefly, PBMC were incubatedwith primary antibodies for 30 minutes at 4° C. Cells were then washedand incubated with Dynabeads coated with goat anti-mouse IgG for 30minutes, before removal of antibody-coated cells by magnetic selection.This process was repeated twice. The negatively selected cells wereroutinely in excess of 98% pure as determined by monoclonal antibodylabeling.

Example 7 Cell Culture and Fibrocyte Differentiation Assay

Cells were incubated in serum-free medium: RPMI (GibcoBRL Life,Invitrogen, Carlsbad, Calif., USA) supplemented with 10 mM HEPES(GibcoBRL/Life), 2 mM glutamine, 100 U/ml penicillin and 100 μg/mlstreptomycin, 0.2% bovine serum albumin (BSA, Sigma), 5 μg/ml insulin(Sigma), 5 μg/ml iron-saturated transferrin (Sigma) and 5 ng/ml sodiumselenite (Sigma). Normal human serum (Sigma), normal human plasma (GulfCoast Regional Blood Center) or fetal calf serum (Sigma), columnfractions, sera and synovial fluid from patients or purified proteinswere added at the stated concentrations. Patient samples were obtainedfrom a repository available to researchers at University of TexasMedical School at Houston. This repository keeps patient informationconfidential, and meets all NIH guidelines.

PBMC were cultured in 24 or 96 well tissue culture plates in 2 ml or 200μl volumes respectively (Becton Dickinson, Franklin Lakes, N.J.) at2.5×105 cells per ml in a humidified incubator containing 5% CO₂ at 37°C. for the indicated times. Fibrocytes in 5 different 900 μm diameterfields of view were enumerated by morphology in viable cultures asadherent cells with an elongated spindle-shaped morphology as distinctfrom small lymphocytes or adherent monocytes. Alternatively cells wereair dried, fixed in methanol and stained with haematoxylin and eosin(Hema 3 Stain, VWR, Houston, Tex.). Fibrocytes were counted using theabove criterion and the presence of an oval nucleus. Enumeration offibrocytes was performed on cells cultured for 6 days in flat-bottomed96 well plates, with 2.5×104 cells per well. In addition, fibrocyteidentity was confirmed by immunoperoxidase staining (see below). Thefibrocyte inhibitory activity of a sample was defined as the reciprocalof the dilution at which it inhibited fibrocyte differentiation by 50%,when added to serum-free medium.

Example 8 Purification and Characterization of Serum and Plasma Proteins

100 ml of frozen human serum or plasma was thawed rapidly at 37° C. and1× “Complete” protease inhibitor (Roche, Indianapolis, Ind., USA), 1 mMbenzamidine HCl (Sigma) and 1 mM Pefabloc (AEBSF:4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride, Roche) wereadded. All subsequent steps were performed on ice or at 4° C. Bariumcitrate adsorption of plasma was performed as described previously. Theprecipitate was collected by centrifugation at 10,000×g for 15 minutes,resuspended in 20 ml of 100 mM BaCl₂ plus inhibitors and recentrifuged.After two rounds of washing, the pellet was resuspended to 20 ml in 10mM sodium phosphate buffer pH 7.4 containing 5 mM EDTA and 1 mMbenzamidine HCl and dialyzed for 24 hours against three changes of 4liters of the same buffer.

Chromatography was performed using an Econo system (Bio-Rad, Hercules,Calif.) collecting 1 ml samples with a flow rate of 1 ml/min. Thedialyzed barium citrate precipitate was loaded onto a 5 ml Hi-TrapHeparin column (Amersham Biosciences) and the column was washedextensively in 10 mM sodium phosphate buffer pH 7.4 until the absorbanceat 280 nm returned to baseline. Bound material was eluted with a steppedgradient of 15 mls each of 100, 200, 300 and 500 mM NaCl in 10 mM sodiumphosphate buffer pH 7.4. The fractions that inhibited monocyte tofibrocyte differentiation eluted at 200 mM NaCl. These were pooled (2ml) and loaded onto a 5 ml Econo-Pak High Q column. After washing thecolumn in 10 mM phosphate buffer, the bound material was eluted with thestepped gradient as above, with the active fraction eluting at 300 mMNaCl.

Active fractions from the High Q chromatography were concentrated to 200μl using Aquacide II (Calbiochem) and then loaded onto a 4-20% nativepolyacrylamide gels (BMA, BioWhittaker, Rockland, Me.) as describedpreviously. After electrophoresis, gel lanes were cut into 5 mm slices,mixed with 200 μl 20 mM sodium phosphate, 150 mM NaCl, 5 mM EDTA pH 7.4containing 1 mM benzamidine HCl, crushed with a small pestle in aneppendorf tube and placed on an end-over-end mixer at 4° C. for 3 days.Proteins that eluted from the gel were analyzed for activity. To obtainamino acid sequences, proteins eluted from the gel slices were loadedonto a 4-20% gel with 100 μM thioglycolic acid in the upper chamber(Sigma). After electrophoresis the gel was rapidly stained with Coomasiebrilliant blue, destained, and the bands excised off the gel. Amino acidsequencing was performed by Dr Richard Cook, Protein SequencingFacility, Department of Immunology, Baylor College of Medicine.

Example 9 Western Blotting

For western blotting, plasma and serum samples were diluted 1:500 in 10mM sodium phosphate pH 7.4. Fractions from heparin and High Q columnswere not diluted. Samples were mixed with Laemmeli's sample buffercontaining 20 mM DTT and heated to 100° C. for 5 minutes. Samples wereloaded onto 4-20% Tris/glycine polyacrylamide gels (Cambrex). Samplesfor native gels were analyzed in the absence of DTT or SDS. Proteinswere transferred to PVDF (Immobilon P, Millipore, Bedford, Mass.)membranes in Tris/glycine/SDS buffer containing 20% methanol. Filterswere blocked with Tris buffered saline (TBS) pH 7.4 containing 5% BSA,5% non-fat milk protein and 0.1% Tween 20 at 4° C. for 18 hours. Primaryand biotinylated secondary antibodies were diluted in TBS pH 7.4containing 5% BSA, 5% non-fat milk protein and 0.1% Tween 20 usingpre-determined optimal dilutions (data not shown) for 60 minutes.ExtrAvidin-peroxidase (Sigma) diluted in TBS pH 7.4 containing 5% BSAand 0.1% Tween 20 was used to identify biotinylated antibody andchemiluminescence (ECL, Amersham Biosciences) was used to visualize theresult.

Example 10 Immunohistochemistry

Cells cultured on 8 well glass microscope slides (Lab-Tek, Nalge NuncInternational, Naperville, Ill.) were air dried before fixation inacetone for 15 minutes. Endogenous peroxidase was quenched for 15minutes with 0.03% H₂O₂ and then non-specific binding was blocked byincubation in 2% BSA in PBS for 60 minutes. Slides were incubated withprimary antibodies in PBS containing 2% BSA for 60 minutes.Isotype-matched irrelevant antibodies were used as controls. Slides werethen washed in three changes of PBS over 15 minutes and incubated for 60minutes with biotinylated goat anti-mouse Ig (BD Pharmingen). Afterwashing, the biotinylated antibodies were detected by ExtrAvidinperoxidase (Sigma). Staining was developed with DAB (Diaminobenzadine,Sigma) for 3 minutes and counterstained for 30 seconds with Mayer'shaemalum (Sigma).

Example 11 Expression of Surface Makers on Fibrocytes

PBMC were cultured in the wells of 8 well glass slides at 2.5×105 cellsper ml (400 μl per well) in serum-free medium for 6 days. Cells werethen air dried, fixed in acetone and stained by immunoperoxidase. Cellswere scored positive or negative for the indicated antigens, compared toisotype-matched control antibodies.

Example 12 Recovery of Protein and Fibrocyte Inhibitory Activity fromFractionated Human Plasma

Plasma was fractionated by BaCl₂ precipitation, heparin and ion exchangechromatography. Protein concentrations were assessed byspectrophotometry at 280 nm. Inhibition of fibrocyte differentiation wasassessed by morphology. The fibrocyte inhibitory activity of a samplewas defined as the reciprocal of the dilution at which it inhibitedfibrocyte differentiation by 50%, when added to serum-free medium.

Example 13 Rat Wound Healing Studies Using High EEO Agarose Bandages

One application of the present invention relates to treatment of smallwounds such as small cuts and surgical incisions as well as chroniculcers, such as diabetic ulcers. Treatments developed for these andsimilar applications may also be readily modified for treatment oflarger wounds and more serious problems.

Local depletion of SAP is important in wound healing and experimentssuch as those described above have revealed that SAP binds particularlywell to a type of agarose known in the art as high EEO agarose. Thisbinding has also been determined to be influenced by the presence ofcalcium.

To test the effects of a calcium/agarose bandage on wound healing, 4 cmwounds through the entire thickness of skin were made on the backs ofthree anesthetized rats. (See FIG. 6.) There was little bleeding fromthe wounds. One rat was treated only with a 4×4 gauze bandage (Topper4×4 sponge gauze, Johnson & Johnson, Skillman, N.J.) lightly soaked with1 ml saline solution (0.9% NaCl w/v in water). This layer of gauze wascovered with a dry 4×4 gauze bandage, and these were held in place withseveral layers of Vetwrap® (3M Animal Care Products, St. Paul, Minn.)which were wrapped around the torso of the rat. A second rat was treatedwith a similar bandage, with the first layer lightly soaked (1 ml) withsaline/5 mM CaCl₂.

A third rat was treated with an agarose/CaCl₂ bandage. To make the firstlayer of this bandage, 0.2 g of high EEO agarose (Electrophoresis gradehigh EEO Agarose product # BP-162, Fisher Scientific, Fair Lawn, N.J.)was dissolved in 20 ml of the saline/CaCl₂ solution described above byheating the solution in a 50 ml polypropylene tube (Falcon, BectonDickinson, Franklin Lakes, N.J.) in a microwave oven until the mixturebegan to boil. After swirling to dissolve the agarose, 1 ml of the hotmixture was poured on a 4×4 gauze bandage that was laying flat on apiece of plastic wrap. The agarose-CaCl₂-saline impregnated gauzebandage was allowed to cool. This was then used as the first layer ofthe bandage for the third rat. A second layer of dry gauze and a coverof Vetwrap® were applied as in the first two rats.

Each rat was separately anesthetized, photographed, and bandaged tominimize differences in time between anesthetizing, wounding andbandaging.

After 24 hours, the rats were lightly anesthetized and weighed, then thebandages were removed and the wounds were photographed. (See FIG. 7.)New bandages of the same initial composition were then reapplied to eachof the rats. After another 24 hours this process was repeated to obtainadditional pictures. (See FIG. 8.)

The rat treated with the agarose/CaCl₂ bandage showed considerably morerapid wound healing than either of the other two rats. (See FIG. 8B.)

Although an agarose bandage was reapplied each day in the presentexample, in other embodiments of the invention an agarose bandage may beapplied only initially or initially and on the first day or so followedby a dry bandage once the wound has substantially closed. Once the woundhas closed, the ability of agarose to absorb SAP may be limited. Woundsthat have closed may also benefit from a dryer environment.

Although hydrated agarose was used in the present example, it may bepossible to also utilize bandages and other formulations with lesshydrated agarose. The agarose may be wetted by serum escaping from thewound itself.

Topical agarose preparations of the present invention may also beprepared using antiseptics to allow both cleansing of the wound andpromotion of wound healing. In a specific example, the agarose may beprepared with alcohol, which may cleanse the wound initially thenevaporate over time.

Example 14 Additional Factors for Use in Topical Wound HealingEmbodiments

Although the above agarose bandages proved quite effective in promotingwound healing, the observed effects can most likely be improved by theaddition of other wound healing factors to the bandages or other topicalagarose formulations. Such factors may include any compound orcompositions, such as small molecules or polypeptides.

In particular, these factors may influence a separate wound healingpathway, or they may influence the fibrocyte formation pathway. They mayalso influence the fibrocyte formation pathway in a different mannerthan SAP, or they may influence it by a mechanism similar to that ofSAP, for example antibodies in the agarose formulation may bind andinactivate additional SAP.

Factors may also be included that address other problems, some of whichmay also affect wound healing. For example, agarose bandages forhemophiliac patients may additionally include clotting factors to helpstop or prevent bleeding from the wound.

In a particular embodiment, IL-4 and/or IL-13 may be included in theagarose formulation. Both are potent activators of the fibroticresponse. IL-4 has been previously described to play a role in woundrepair and healing.

Experiments have shown that IL-4 and IL-13 are capable of promotingfibrocyte differentiation in vitro. Specifically, PBMC were cultured inserum-free medium in the presence of IL-4 or IL-13. Concentrations ofeither IL-4 or IL-13 between 10 and 0.1 ng/ml enhanced the number offibrocytes in culture. (See FIG. 9.) This indicates that IL-4 and IL-13are capable of promoting the differentiation of fibrocyte precursorsinto mature fibrocytes. Therefore a bandage or other topical agaroseformulation as described above additionally containing IL-4 and/or IL-13is expected to show further improvements in wound healing.

Other factors that may be added to agarose bandages or topicalformulations as described above or physiological conditions to bemimicked include:

Molecules known to bind to SAP:

-   -   Collagen IV;    -   Laminin;    -   Fibronectin;    -   C4BP;    -   Aggregated Fc of IgG;    -   CD16, CD32 and CD64: Fc Receptors;    -   Heparin;    -   LPS;    -   Apoptotic cells, especially chromatin and DNA    -   Zymozan.

Physiological conditions related to SAP binding:

-   -   SAP exhibits calcium-dependent binding to amyloid fibers formed        from, e.g. serum amyloid A (SAA), immunoglobulin light chains,        β2 microglobulin, transthyretin and the neurofibrillary tangles;    -   SAP binds to surfaces of bacteria due to expression of pyruvate        acetyl of galactose and to other sugars on the surface of        bacteria.    -   SAP binds to the “artificial” ligands on high EEO agarose and        phosphoethanolamine-agarose, and with low affinity to        phosphocholine-sepharose. These features give rise to two ways        of purifying SAP from serum or plasma. First, SAP may be bound        to high EEO agarose via the pyruvate acetyl of galactose, which        is a minor constituent of agarose preparations. Second, SAP may        be bound to phosphoethanolamine-agarose, which is presently the        preferred method of SAP purification in the art. Thus,        phosphoethanolamine-agarose may be used for bandages or topical        formulations.

Example 15 Methods of Identifying Suitable SAP-Binding Agents IncludingDerivatized Agarose

Because the biological function of SAP includes opsonization of foreignmolecules for enhanced uptake by macrophages, other derivitized agarosesincorporating motifs such as bacterial cell wall carbohydrates, DNA orDNA analogs, and the like may also be used if they meet the followingcriterion for activity.

Prepare a 100 microliter sample of SAP at 20 micrograms/milliliter. Addinsoluble adsorbent in an amount that increases the volume of the sampleby less than 100%. Incubate with gentle shaking or end over end rotationfor 1 hour. Centrifuge to pellet the adsorbent. Measure remaining SAP inthe supernatant. If more than 50% of the SAP has been removed, theadsorbent is deemed active.

The methodology may also be used to identify and test other SAP-bindingagents.

Example 16 Pig Wound Healing Studies Using Agarose Hydrogels

The effects of agarose hydrogels on deep partial thickness wound healingin a porcine model were also studied. The porcine model hasmorphological similarities to human skin. A total of seven young femalespecific pathogen free (SPF: Ken-O-Kaw Farms, Windsor, Ill.) pigsweighing 25-30 kg were maintained in constant conditions for two weeksprior to the experiment. These animals were fed a basal diet ad libitumand were housed individually in animal facilities in compliance with theAmerican Association for Accreditation of Laboratory Animal Care withcontrolled temperature (19-21° C.) and lighting (12 hours light/12 hoursdark).

The flank and back of the experimental animals were clipped withstandard animal clippers on the day of the experiment. The skin on bothsides of each animal was prepared for wounding by washing with anon-antibiotic soap (Neutrogena Soap Bar; Johnson & Johnson, Calif.) andsterile water. Each animal was anesthetized intramuscularly withtiletamine HCl plus zolazepam (1.4 mg/kg) (Telazol; Laderle PatenteralsInc., Puerto Rico), xylazine (2.0 mg/kg) (X-jet; Phoenix ScientificInc., MO), and atropine (0.04 mg/kg) (Atrojet SA, Phoenix ScientificInc., MO) followed by mask inhalation of isoflurane (Isothesia, AbbottLaboratories, IL) and oxygen combination.

One hundred and sixty (160) rectangular wounds measuring 10 mm×7 mm×0.5mm were made in the paravertebral and thoracic area with a specializedelectrokeratome fitted with a 7 mm blade. The wounds were separated fromone another by 15 mm of unwounded skin.

Forty wounds were randomly assigned to a treatment group according toone the three experimental designs. One animal in a preliminary studywas assigned to a treatment group where wounds received either i) MEAgarose gel, ii) SP Agarose gel, ii) the vehicle alone, or iv) wereuntreated and exposed to air. Both ME Agarose ans SP Agarose gels metthe criteria stated in Example 15.

One other animal in the preliminary study was assigned to a treatmentgroup where wounds received either i) SP Agarose gel, ii) the vehiclealone, iii) Vigilon wound dressing (C.R. Bard, Inc., GA) or iv) wereuntreated and exposed to air.

For the preliminary study, three animals were included. In theseexperiments two hydrogel test agents (SP and ME Agarose) along withpositive and negative controls were evaluated. These treatments wererandomized among these three animals with two of the animals receivingSP Agarose hydrogel material. Because it appeared that the SP materialwas more effective than the ME Agarose hydrogel, four additional animalswere studied using the SP Agarose hydrogel alone.

Four animals were assigned to a treatment group where wounds receivedeither i) SP Agarose gel, ii) the vehicle alone, iii) Vigilon, or iv)were untreated and exposed to air. All wounds in all treatment groupswere covered with a polyurethane dressing except those that wereuntreated an exposed to air.

The application and assessment of different treatment groups is shown inFIG. 10. Areas A, B, C, and D are repeated areas of treatment.

All hydrogel treated wounds were treated by placing the hydrogel mateialover the wounds and surrounding normal skin to the approximate thicknessof the Vigilon (˜1 mm). The hydrogel was then covered with apolyurethane dressing to prevent desiccation. One day 1 after treatment,the animals were anesthetized and the dressings observed to make surethey were still intact. All materials were kept in place until woundevaluation unless it was observed that the materials needed to bereplaced. In order to assess the wounds, a portion of the hydrogel wasremoved to uncover five wounds for evaluation. Wounds were evaluated forepithelization as described below.

Animals were monitored daily for any observable signs of pain ordiscomfort. In order to help minimize possible discomfort, an analgesicbuprenorphine 0.03 mg/kg (Buprenex injectable, Reckitt Benckiser Hull,England) was given to each animal on the first day, and every third daythereafter, while under anesthesia. A fentanyl transdermal system: 25μg/hr (Duragesic; Alza Corp., CA) was used during the entire experiment.

Wounds were examined regularly for any signs of erythema (redness) andinfection. The physical characteristics of the material were also noted.

Beginning on day 3 after wounding for the first 3 pigs in thepreliminary study and on day 4 after wounding for the final 4 pigs andon each day thereafter until the time of healing (day 6 for mosttreatment groups), five wounds and the surrounding normal skin from eachtreatment group were excided using an electrokeratome with a 22 mm bladeset to a depth of 0.7 mm. (See FIG. 10.) All specimens that were notexcised intact were discarded. The excised skin containing the woundsite was inclubated in 0.5 M sodium bromide at 37° C. for 24 hours,allowing for a separation of the dermis from the epidermis. Afterseparation, the epidermal sheet was examined macroscopically fordefects. (FIG. 11.) Defects were defined as holes in the epidermal sheetor as a lack of epidermal continuity in the area of the wound.Epithelization was considered complete (healed) if no defect(s) werepresent; any defect in the wound area indicates that healing isincomplete.

The hydrogel materials did not cause any re-injury of wounds duringremoval throughout the entire assessment time. During the laterevaluations (days 8-10) the hydrogel materials appeared to decrease inthickness (density) and became moderately desiccated, forming aglue-like substance.

The untreated air exposed wounds displayed prominent crust formation ascompared to wounds from the other treatment groups. None of the woundsfrom any of the treatment groups showed signs of erythema or infection.Wounds treated with all hydrogel materials appeared to havesignificantly less crust formation as compared to wounds in theuntreated air exposed group.

After the study was completed the number of wounds completely healed(completely epithelized) was divided by the total number of woundssampled per day and multiplied by 100 to detrmine the % epilthelization(FIG. 12).

The Chi square test was used to determine statistical significancebetween the treatment groups. All data show in FIG. 12 varied in astatistically significant manner based on this test.

Specific results by day were also examined. On day 3 none of the woundsin any treatment group were healed.

On day 4, 33% of the wounds treated with the SP Agarose hydrogel werehealed. 13% of the wounds treated with the vehicle were healed. None ofthe other wounds were healed on that day.

On day 5, 90% of the wounds treated with SP Agarose gel were completelyre-epithelized and 80% of the wounds in the vehicle group were healed.Wounds in the Vigilon group were 56% healed and 3% of the wounds in theuntreated group were healed.

On day 6, 100% of the wounds treated with SP Agarose gel, Vigilon andthe vehicle were healed. Only 40% of the untreated wounds were healed.

On day 7, 100% of the wounds from all treatment groups were completelyre-epithelized except those from the untreated group, which were 60%healed.

On day 8, all wounds from each treatment group were completelyre-epithelized.

These results collectively show that all hydrogel treatment groupsincreased the rate of epithelization as compared to untreated,air-exposed control wounds. These treatment groups initiated 100%complete epithelization two days earlier than the untreated, air-exposedwounds.

Further, wounds treated with the SP Agarose hydrogel healedsignificantly faster than wounds treated with Vigilon.

Although only exemplary embodiments of the invention are specificallydescribed above, it will be appreciated that modifications andvariations of these examples are possible without departing from thespirit and intended scope of the invention.

1. A wound dressing comprising: a Serum Amyloid P (SAP)-binding agarose,the SAP-binding agarose operable to promote healing of a skin injury orlaceration in a mammal; and a divalent cation in an amount sufficient topromote healing of the skin injury or laceration in conjunction with theSAP-binding agarose in a concentration sufficient to promote healing ofthe skin injury or laceration more quickly than the SAP-binding agarosealone.
 2. The wound dressing of claim 1, further comprising anadditional wound healing factor.
 3. The wound dressing of claim 2,wherein the additional wound healing factor is selected from the groupconsisting of: interleukin (IL)-4, IL-13, fibroblast growth factor(FGF), transforming growth factor beta (TGFβ), and any combinationsthereof.
 4. The wound dressing of claim 1, further comprising IL-13 at aconcentration of 0.1 to 10 ng/ml.
 5. The wound dressing of claim 1,further comprising IL-4 at a concentration of 0.1 to 10 ng/ml.
 6. Thewound dressing of claim 1, further comprising a bandage.
 7. The wounddressing of claim 1, wherein the divalent cation is Ca²⁺.
 8. A wounddressing comprising: a Serum Amyloid P (SAP)-binding highelectroendosmosis (EEO) agarose, the SAP-binding agarose operable topromote healing of a skin injury or laceration in a mammal; and adivalent cation in an amount sufficient to promote healing of the skininjury or laceration in conjunction with the SAP-binding agarose in aconcentration sufficient to promote healing of the skin injury orlaceration more quickly than the SAP-binding agarose alone.
 9. The wounddressing of claim 8, wherein the divalent cation is Ca²⁺.
 10. The wounddressing of claim 9, further comprising approximately 1% (w/v) high EEOagarose.
 11. The wound dressing of claim 8, further comprising anadditional wound healing factor.
 12. The wound dressing of claim 11,wherein the additional wound healing factor is selected from the groupconsisting of: interleukin (IL)-4, IL-13, fibroblast growth factor(FGF), transforming growth factor beta (TGFβ), and any combinationsthereof.
 13. The wound dressing of claim 12, further comprising IL-13 ata concentration of 0.1 to 10 ng/ml.
 14. The wound dressing of 12,further comprising IL-4 at a concentration of 0.1 to 10 ng/ml.
 15. Thewound dressing of claim 8, further comprising a bandage.
 16. The wounddressing of claim 8, wherein the SAP-binding high EEO agarose comprisesa pyruvate acetal of galactose.
 17. A wound dressing comprising: a SerumAmyloid P (SAP)-binding agarose comprising a phosphoethanolamine moiety,the SAP-binding agarose operable to promote healing of a skin injury orlaceration in a mammal; and a divalent cation in an amount sufficient topromote healing of the skin injury or laceration in conjunction with theSAP-binding agarose in a concentration sufficient to promote healing ofthe skin injury or laceration more quickly than the SAP-binding agarosealone.
 18. The wound dressing of claim 17, wherein the divalent cationis Ca²⁺.
 19. The wound dressing of claim 17, further comprising anadditional wound healing factor.
 20. The wound dressing of claim 19,wherein the additional wound healing factor is selected from the groupconsisting of: interleukin (IL)-4, IL-13, fibroblast growth factor(FGF), transforming growth factor beta (TGFβ), and any combinationsthereof.
 21. The wound dressing of claim 17, further comprising IL-13 ata concentration of 0.1 to 10 ng/ml.
 22. The wound dressing of claim 17,further comprising IL-4 at a concentration of 0.1 to 10 ng/ml.
 23. Thewound dressing of claim 17, further comprising a bandage.